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UNIVERSITY of CALIFORNIA, SAN DIEGO the Intricacies of UGT UNIVERSITY OF CALIFORNIA, SAN DIEGO The Intricacies of UGT Regulation: Protein-Protein Interactions and Environmental Arsenic Exposure A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Chemistry by Camille Maria Konopnicki Committee in charge: Professor Robert H. Tukey, Chair Professor Pieter Dorrestein Professor James Halpert Professor Alexander Hoffmann Professor William Trogler 2012 Copyright Camille Maria Konopnicki, 2012 All rights reserved. The Dissertation of Camille Maria Konopnicki is approved, and it is acceptable in quality and form for publication on microfilm and electronically: _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ Chair University of California, San Diego 2012 iii DEDICATION To my parents, Marek Konopnicki and Barbara Pawlowski-Konopnicki and my family, past and present. iv EPIGRAPH Play is the highest form of research. Albert Einstein v TABLE OF CONTENTS SIGNATURE PAGE ................................................................................iii DEDICATION .......................................................................................... iv EPIGRAPH ................................................................................................ v TABLE OF CONTENTS .......................................................................... vi LIST OF ABBREVIATIONS .................................................................viii LIST OF FIGURES.................................................................................. xii LIST OF TABLES .................................................................................. xiv ACKNOWLEDGEMENTS ..................................................................... xv VITA ........................................................................................................ xx ABSTRACT OF THE DISSERTATION..............................................xxiii CHAPTER 1 Introduction to Drug Metabolism ........................................ 1 Drug Metabolizing Enzymes from an Evolutionary Perspective...................................2 The Function of Phase I and Phase II Drug Metabolizing Enzymes..............................3 Characterization of the Glucuronidation Process...........................................................4 UDP-Glucuronsyltransferases ........................................................................................6 Tissue-Specific Expression of the UGTs .....................................................................12 Individual UGT Variability, Disease, and Adverse Drug Reactions............................14 Protein-Protein Interactions Between DMEs ...............................................................20 UGT-UGT Interactions ................................................................................................22 RNA Interference as a Tool to Study Glucuronidation................................................24 The Xenobiotic Receptors ............................................................................................27 The Aryl Hydrocarbon Receptor..................................................................................29 Nuclear Receptors ........................................................................................................33 The Pregnane X Receptor and the Constitutive Androstane Receptor.................................37 The Oxidative Stress Sensor, NF-E2 related factor-2 ..................................................42 The UGT1A1 Phenobarbital Response Enhancer Module ...........................................49 The NF-κB/IKK Pathway.............................................................................................50 The Mitogen Activated Protein Kinases.......................................................................55 vi Objectives of the Dissertation ......................................................................................61 References ....................................................................................................................65 CHAPTER 2 Evaluation of UGT Protein Interactions in Human Hepatocytes: Effect of siRNA Down Regulation of UGT1A9 and UGT2B7 on Propofol Glucuronidation in Human Hepatocytes ............ 112 Introduction ................................................................................................................113 Experimental...............................................................................................................115 Results ........................................................................................................................120 Propofol Glucuronidation in Recombinant UGTs .....................................................120 siRNA Characterization .............................................................................................121 Inhibition of Propofol Glucuronidation by siRNA Down Regulation in Human Hepatocytes ................................................................................................................127 Discussion...................................................................................................................128 References ..................................................................................................................132 CHAPTER 3 The Regulatory Role of Oral Arsenic in hUGT1 Mice ... 137 Introduction ................................................................................................................138 Humanized Mice as a Sensor for Environmental Toxicant Exposure .......................138 Environmental Arsenic Contamination......................................................................140 Arsenic-Induced Generation of Reactive Oxygen Species ........................................143 Arsenic Can Modulate Xenobiotic Transcriptional Activation..................................146 Arsenic Impacts the NF-κB/IKK Signaling Pathway ................................................149 Arsenic Influences the MAPK Signaling Pathway ....................................................152 Cell Cycle Dysregulation and Morphological Changes Occur with Arsenic Exposure ....................................................................................................................................156 Experimental...............................................................................................................161 Results ........................................................................................................................166 Intestinal UGT1A1 Induction Occurs with Oral Arsenic Exposure ...........................166 CAR is not Involved in Regulating Arsenic-Induced UGT1A1 Expression ..............167 The NF-κB/IKK Pathway is not Involved in Regulating Intestinal UGT1A1............171 Oxidative Stress Induced Nrf2 Activation Upregulates UGT1A1 Expression...........172 UGT1A1 Induction Occurs Independent of MAPK Activation .................................175 Arsenic Exposure Causes Intestinal Damage, Changes in Cellular Morphology, and Increases Proliferation................................................................................................176 Discussion...................................................................................................................180 References ..................................................................................................................188 CHAPTER 4 General Conclusions........................................................ 202 Future Implications of this Work ...............................................................................203 UGT Interactions in Human Hepatocytes ..................................................................203 Intestinal Microflora and UGT1A1 Induction ............................................................206 References ..................................................................................................................208 vii LIST OF ABBREVIATIONS ADRs adverse drug reactions AhR aryl hydrocarbon receptor ARE antioxidant response element ARNT aryl hydrocarbon receptor nuclear translocator As3+ arsenite, inorganic trivalent arsenic As5+ arsenate, inorganic pentavalent arsenic B[a]P benzo[a]pyrene CAR constitutive androstane receptor CAT catalase CN-I,II Crigler-Najjar syndrome type I or II COX-2 cyclooxygenase-2 CPT-11 irinotecan CYP cytochrome P450 DME drug metabolizing enzyme dsRNA double-stranded RNA ER endoplasmic reticulum ERK extracellular signal-related kinase G6PD glucose-6-phosphate dehydrogenase Gadd45β growth arrest and DNA-damage-inducible gene 45β GI gastrointestinal tract GPx glutathione peroxidase GR glucocorticoid receptor viii GSH-Re glutathione reductase GST glutathione S-transferase GSTA1,2 glutathione S-transferase A1 or 2 HCC hepatocellular carcinoma H&E hematoxylin and eosin HLM human liver microsomes HO-1 heme oxygenase-1 HPLC-MS/MS high-performance liquid chromatography/tandem mass spectrometry HRE hormone response element hUGT1 humanized UGT1 iAs inorganic arsenic IKK IκB kinase JNK c-Jun NH2-terminal protein kinase Keap1 Kelch-like ECH-associated protein 1 Km Michaelis-Menten constant;
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